Analysis And Identification Of MiRNAs And Their Target Genes In Silver Carp (Hypophthalmichthys Molitrix) Under Hypoxia Stress

Silver carp(Hypophthalmichthys molitrix)is an important economic fish in China,which is favored by consumers.It is widely distributed in the natural water,which plays an important role in regulating the ecological environment.In the process of culture of silver carp,hypoxia has become an important factor restricting the increase of its yield.Hypoxia is one of the most serious stressors in silver carp culture.Too low oxygen content in the water leads to changes in the physiology or body of fish,and serious hypoxia even result in death of fish.micro RNA(miRNA)is a kind of endogenous non-coding single-stranded small RNA,which exists widely in all kinds of animals and plants,and plays a key role in cell development,immune response and the regulation of some genes.In this study,liver,brain,heart and gill of silver carp under hypoxia stress were collected to construct RNA mixed pool.Small RNA high-throughput sequencing was carried out by Illumina Noveseq sequencing platform,and then bioinformatics analysis was carried out to predict target gene and analyze the GO and KEGG enrichment.Differential expression of miRNA was screened and analyz furtherly.Furthermore,the target genes of differentially expressed miRNA were predictedand verified by dual-luciferase reporter technique.The main results are as follows:1 Sequencing of small RNA Library of silver carp under hypoxia stressIn order to explore the expression and regulation of miRNA in silver carp under different degrees of hypoxia stress,silver carp were divided into normoxia group(T0),hypoxia group(T1),semi-asphyxia group(T2)and asphyxia group(T3).Silver carp individuals was sampled to construct the small RNA library and high-throughput sequencing.The results showed that 26475225,26143905,22941194 and 28984723 small RNA Raw reads were obtained from T0,T1,T2 and T3 respectively,and25558042,25542062,19466537 and 28030238 valid sequences(Clean read)were obtained base on screening the original data,respectively.After analyzing the obtained small RNA length,it is found that most of the small RNA length is 21-24 nt,which accords with the length distribution range of miRNA.By comparing with miRbase(V22)database,299 known miRNAs were identified,which 288,286,249 and 280 miRNAs were matched in T0,T1,T2 and T3 groups,respectively.Based on the base preference analysis of the identified miRNA,it was found that A base and U base accounted for the most in each group.Subsequently,miREvo and miRdeep software were used to predict novel miRNAs,and a total of 391 novel miRNAs were predicted,305,279,165 and 298 miRNAs were predicted by T0,T1,T2 and T3,respectively.The sequencing data provided a basis for exploring the expression of miRNA in silver carp under different levels of hypoxia stress.2 Identification of differentially expressed miRNA and target genesDifferential expression analysis was performed on known miRNA and novel miRNA by edge R software,and pairwise comparison was performed on T0 and T1,T0 and T2,T0 and T3,T1 and T3,and T2 and T3.The number of differentially upregulated miRNAs was 75,210,104,183,100 and 144,respectively.The number of down-regulated differential miRNAs was 55,112,60,152,95 and 186,respectively.Then the target genes of these differentially expressed miRNA were predicted by Miranda and q Tar software.The results of the intersection of the two software were selected,and the predicted target genes were further analyzed by GO and KEGG enrichment analysis.The results showed that the target genes were mainly enriched in Signal transduction,Cell growth and death,Immune system,Lipid metabolism and other pathways.The results showed that the differentially expressed miRNA target genes were related to apoptosis,immunity and energy metabolism of silver carp under hypoxia stress.Ten differentially expressed miRNA were randomly selected from each control group for q PCR identification.The results showed that the expression trend of differentially expressed miRNA based on q PCR was basically consistent with that in the sequencing results,which demonstrates the high accuracy of the sequencing data.3 Target gene verification and expression pattern of miR-17a-5p under hypoxia stressIn order to further explore the relationship between the differentially expressed miRNA and the target genes,the target genes of miR-17a-5p were predicted by Miranda and q Tar software.The results showed that there was a targeted relationship between miR-17a-5p and hypoxia inducible factor-1 α(HIF-1 α).The targeting relationship between w T-pmir GLO-HIF-1α 3’ UTR and mutated vector MUt-Pmirglo-HIF-1α 3’ UTR were successfully cloned using the dual-luciferase reporting technique.293 T cells were transfected with miR-17a-5p mimics of miR-17a-5p and negative control mimics NC,respectively,and lucifase activity was detected 48 h later.The results showed that the luciferase activity decreased significantly in the combination of miR-17a-5p mimics and WT-pmir GLO-HIF-1α 3’UTR,but there was no significant change in other combinations.It was proved that HIF-1α was a potential target gene of miR-17a-5p.In order to understand the expression pattern of miR-17a-5p,the expression of miR-17a-5p in liver,heart,brain and gill of silver carp under different levels of hypoxia stress was identified by q PCR.The results showed that the expression of miR-17a-5p in gill was higher than that in the other three tissues,and the expression level of miR-17a-5p in hypoxia group and semi-asphyxiated group was significantly higher than that in normoxic group and asphyxia group.